The long-term objective is to understand the operation of metal-ion clusters and electron/proton transfer processes in biology. The specific goal is to understand the mechanism of photosynthetic water oxidation. Oxygenic photosynthesis provides the molecular oxygen and fixed carbon required to sustain all animal life. The proposed work will identify amino acid residues that coordinate the manganese and calcium ions at the catalytic site of water oxidation or whose protonation properties influence the reactivity of the manganese cluster. Identification of these residues will impose new constraints on models for the operation of the manganese cluster, and will enable such models to be placed in the context of protein structure. The information obtained will be applicable to other metal-ion clusters, and to the study of membrane proteins and biological electron and proton transfer processes in general. Electron and proton transfer reactions can be studied more easily and precisely in photosynthetic than in mitochondrial systems because both processes can be conveniently initiated with light. In oxygenic photosynthesis, Photosystem II (pSII) uses light to extract electrons from water and donate them into an electron transport chain that generates the chemical free energy and reducing equivalents required for carbon fixation. PSII photochemistry takes place in a heterodimer of two polypeptides known as D1 and D2. A cluster of four manganese ions accumulates four oxidizing equivalents in response to this photochemistry, then oxidizes two molecule of water by a process that requires calcium and releases molecular oxygen as a by-product. The proposed work will (l) determine if specific amino acid residues of the D1 polypeptide (e.g, Asp- 170, His-332, Glu-333, His-337, and Asp-342) ligate the Mn cluster, (2) characterize the role of specific amino acid residues of the D1 polypeptide in the binding of calcium (e.g., Asp-59, Asp-61, His-332, Glu- 333, His-337, and Asp-342), (3) further characterize the influence of particular residues of the D1 polypeptide on the operation of the intact Mn cluster (e.g., Asp61, His-92, and Glu-189), and (4) identify amino acid residues whose protonation properties influence the energetics and mechanism of water oxidation. Mutants with substitutions of the latter residues would contain Mn clusters that are significantly perturbed or are non-functional and that exhibit altered patterns of proton release. The required mutations have already been constructed by us in the cyanobacterium Synechocystis sp. PCC 6803.